Kidney injury molecule-<em>1</em> (Kim-<em>1</em>) is associated with ischemic and proteinuric tubular injury; however, whether dysregulation of the renin-<em>angiotensin</em> system (RAS) can also induce Kim-<em>1</em> is unknown. We studied Kim-<em>1</em> expression in homozygous Ren2 rats, characterized by renal damage through excessive RAS activation. We also investigated whether antifibrotic treatment (RAS blockade or p38 MAP kinase inhibition) would affect Kim-<em>1</em> expression. At <em>7</em> wk of age, homozygous Ren2 rats received a nonhypotensive dose of candesartan (0.05 mg x kg(-<em>1</em>) x day(-<em>1</em>) sc) or the p38 inhibitor SB-239063 (<em>1</em>5 mg x kg(-<em>1</em>) x day(-<em>1</em>) sc) for 4 wk; untreated Ren2 and Sprague-Dawley (SD) rats served as controls. Kim-<em>1</em> mRNA and protein expression were determined by quantitative PCR and immunohistochemistry, respectively, and related to markers of prefibrotic renal damage. Urinary Kim-<em>1</em> was measured in 8-wk-old Ren2 and SD rats with and without <em>angiotensin</em>-converting enzyme inhibition (ramipril, <em>1</em> mg x kg(-<em>1</em>) x day(-<em>1</em>) in drinking water for 4 wk). Untreated Ren2 rats showed a >20-fold increase in renal Kim-<em>1</em> mRNA (expressed as Kim-<em>1</em>-to-GAPDH ratio): <em>7</em>5.5 +/- 43.6 vs. 3.<em>1</em> +/- <em>1</em>.0 in SD rats (P < 0.0<em>1</em>). Candesartan and SB-239063 strongly reduced Kim-<em>1</em> mRNA: 3.<em>1</em> +/- <em>1</em>.5 (P < 0.0<em>1</em>) and 9.8 +/- 4.2 (P < 0.05), respectively. Kim-<em>1</em> protein expression in damaged tubules paralleled mRNA expression. Kim-<em>1</em> expression correlated with renal osteopontin, alpha-smooth muscle actin, and collagen III expression and with tubulointerstitial fibrosis. Damaged tubular segments expressing activated p38 also expressed Kim-<em>1</em>. Urinary Kim-<em>1</em> was increased in Ren2 vs. SD (458 +/- <em>7</em>0 vs. 2<em>7</em> +/- 2 pg/ml, P < 0.0<em>1</em>) rats and abolished in Ren2 rats treated with ramipril (33 +/- 5 pg/ml, P < 0.0<em>1</em>). Kim-<em>1</em> is associated with development of RAS-mediated renal damage. Antifibrotic treatment through RAS blockade or p38 MAP kinase inhibition reduced Kim-<em>1</em> in the homozygous Ren2 model.

We studied the cellular distribution of <em>angiotensin</em>-converting enzyme (ACE) in the heart related to the cell types involved in left ventricular repair and remodeling before and after myocardial infarction by immunohistochemical techniques using monoclonal and polyclonal antibodies. In noninfarcted myocardium of both human and rat, ACE expression was confined to endothelial cells and subendocardial cell layers of the aortic valve. ACE was prominent in endothelia of small arteries and arterioles, whereas only half the coronary capillaries were immunoreactive and venous vessels were almost completely devoid of the enzyme. In a rat model of myocardial infarction, ACE distribution was determined <em>1</em>, 3, and <em>7</em> days and 2, 3, and 6 weeks after coronary occlusion. Three and <em>7</em> days after infarction, endothelial cells of sprouting capillaries and macrophages in the marginal zone of necrosis revealed ACE expression. In both human and rat with the onset of fibrosis, intense staining of the enzyme was found in the marginal zone of the repair tissue. In situ hybridization for collagen type I in the rat revealed that zones with high collagen content had almost no ACE immunoreactivity. Vascular smooth muscle cells and cardiomyocytes revealed no ACE expression throughout the study. We conclude that endothelial cells are the principal source for the expression of ACE after myocardial infarction. The observed induction of ACE with the onset of fibrosis suggests a role of this enzyme that is related to tissue repair and remodeling.

Plasma levels of atrial natriuretic peptide (ANP), <em>angiotensin</em> II, aldosterone, and catecholamines were followed for <em>1</em> month and then for 4 to 6 months in 55 patients with acute myocardial infarction. Plasma hormones were highest within the first 24 hours after the onset of infarction but normalized during the first few days in patients without heart failure. In patients with symptoms of heart failure, <em>angiotensin</em> II and norepinephrine remained elevated for <em>1</em> month and ANP remained elevated for 4 to 6 months. During head-up tilt, <em>angiotensin</em> II and norepinephrine increased most in patients with overt heart failure. Among patients with a first myocardial infarction, a positive correlation was found between infarct size and ANP, <em>angiotensin</em> II, and norepinephrine on day 5 to <em>7</em> and between infarct size and <em>angiotensin</em> II during head-up tilt at <em>1</em> month, and between infarct size and ANP at <em>1</em> month. A similar relationship was found when only patients without heart failure were studied. It is concluded that sustained neurohormonal activation after myocardial infarction mainly occurs in patients with clinical heart failure but is related to the magnitude of myocardial damage, even in patients without heart failure. Measurement of neurohormones during head-up tilt may be an additive assessment for the detection of neurohormonal activation.

Arterial baroreflexes are well established to provide the basis for short-term control of arterial pressure; however, their role in long-term pressure control is more controversial. We proposed that if the sustained decrease in renal sympathetic nerve activity (RSNA) we observed previously in response to <em>angiotensin</em> II-induced hypertension is baroreflex mediated, then the decrease in RSNA in response to <em>angiotensin</em> II would not occur in sinoaortic-denervated (SAD) animals. Arterial pressure and RSNA were recorded continuously via telemetry in sham and SAD rabbits living in their home cages before, during, and after a <em>7</em>-day infusion of <em>angiotensin</em> II (50 ng . kg(-<em>1</em>) . min(-<em>1</em>)). The arterial pressure responses in the 2 groups of rabbits were not significantly different (82+/-3 mm Hg sham versus 83+/-3 mm Hg SAD before <em>angiotensin</em> II infusion, and <em>1</em>0<em>1</em>+/-6 mm Hg sham versus <em>1</em>00+/-4 mm Hg SAD day 6 of <em>angiotensin</em> II). In sham rabbits, there was a significant sustained decrease in RSNA (53+/-<em>7</em>% of baseline on day 2 and 65+/-<em>7</em>% on day 6 of the <em>angiotensin</em> II). On ceasing the <em>angiotensin</em> II, all variables recovered to baseline. In contrast, RSNA did not change in SAD rabbits with the <em>angiotensin</em> II infusion (RSNA was 98+/-8% of baseline on day 2 and 98+/-8% on day 6 of the <em>angiotensin</em> II infusion). These results support our hypothesis that the reduction in RSNA in response to a pressor dose of <em>angiotensin</em> II is dependent on an intact arterial baroreflex pathway.

<em>1</em>. The pharmacological profile of valsartan, (S)-N-valeryl-N-([2'-(<em>1</em>H-tetrazol-5-yl)biphenyl-4-yl]-methyl)-vali ne, a potent, highly selective, and orally active antagonist at the <em>angiotensin</em> II (AII) AT<em>1</em>-receptor, was studied in vitro and in vivo. 2. Valsartan competed with [<em>1</em>25I]-AII at its specific binding sites in rat aortic smooth muscle cell membranes (AT<em>1</em>-receptor subtype) with a Ki of 2.38 nM, but was about 30,000 times less active in human myometrial membranes (AT2-receptor subtype). 3. In rabbit aortic rings incubated for 5 min with valsartan, at concentrations of 2, 20 and 200 nM, the concentration-response curve of AII was displaced to the right and the maximum response was reduced by 33%, 36% and 40%, respectively. Prolongation of the incubation time with valsartan to <em>1</em> h or 3 h, further reduced the maximum response by 48% or 59% (after 20 nM) and by 59% or 60% (after 200 nM) respectively. After 3 h incubation an apparent pKb value of 9.26 was calculated. Contractions induced by noradrenaline, 5-hydroxytryptamine, or potassium chloride were not affected by valsartan. No agonistic effects were observed in the rabbit aorta at concentrations of valsartan up to 2 microM. 4. In bovine adrenal glomerulosa, valsartan inhibited AII-stimulated aldosterone release without affecting the maximum response (pA2 8.4). 5. In the pithed rat, oral administration of valsartan (<em>1</em>0 mg kg-<em>1</em>) shifted the AII-induced pressor response curves to the right, without affecting responses induced by the electrical stimulation of the sympathetic outflow or by noradrenaline. Animals treated with valsartan 24 h before pithing also showed significant inhibition of the response to AII. 6. In conscious, two-kidney, one-clip renal hypertensive rats (2K<em>1</em>C), valsartan decreased blood pressure in a dose-dependent manner after single i.v. or oral administration. The respective ED30 values were 0.06 mg kg-<em>1</em> (i.v.) and <em>1</em>.4 mg kg-<em>1</em> (p.o.). The antihypertensive effect lasted for at least 24 h after either route of administration. After repeated oral administration for 4 days (3 and <em>1</em>0 mg kg-<em>1</em> daily), in 2K<em>1</em>C renal hypertensive rats, systolic blood pressure was consistently decreased, but heart rate was not significantly affected. <em>7</em>. In conscious, normotensive, sodium-depleted marmosets, valsartan decreased mean arterial pressure, measured by telemetry, after oral doses of <em>1</em>-30 mg kg-<em>1</em>. The hypotensive effect persisted up to <em>1</em>2 h after 3 and <em>1</em>0 mg kg-<em>1</em> and up to 24 h after 30 mg kg-<em>1</em>. 8. In sodium-depleted marmosets, the hypotensive effect of valsartan lasted longer than that of losartan(DuP <em>7</em>53). In renal hypertensive rats, both agents had a similar duration (24 h), but a different onset of action (valsartan at <em>1</em> h, losartan between 2 h and 24 h).9. These results demonstrate that valsartan is a potent, specific, highly selective antagonist of AII at theAT<em>1</em>-receptor subtype and does not possess agonistic activity. Furthermore, it is an efficacious, orally active, blood pressure-lowering agent in conscious renal hypertensive rats and in conscious normotensive,sodium-depleted primates.

Here we demonstrated, by RT-PCR analysis, the expression of both <em>angiotensin</em> II (Ang II) receptor subtypes, AT<em>1</em> and AT2, in a breast cancer epithelial cell line, MCF-<em>7</em>. Ang II was not able to affect the intracellular Ca2+ concentration in Fura-2 loaded cells suggesting that AT<em>1</em>-mediated phospholipid hydrolysis is not involved in its intracellular transduction pathway. Ang II modulated the activity of the Na+/K+ATPase in a dose- and time-dependent manner and was mitogenic, with a dose-dependent (<em>1</em>-<em>1</em>000 nM) proliferative effect and a maximal response at <em>1</em>00 nM. Both Na+/K+ATPase activation and stimulation of proliferation were mediated by binding of Ang II to AT<em>1</em>, as the effects were completely blocked by DuP <em>7</em>53, a specific AT<em>1</em> antagonist. CGP 42<em>1</em><em>1</em>2, an AT2 antagonist, did not affect Ang II actions. The main conclusion of this study is that Ang II exerts its effects on cell proliferation and Na+/K+ATPase in breast cancer epithelial cells, MCF-<em>7</em>, via AT<em>1</em> activation independently of the Ca(2+) signalling mechanism.

A total of 3<em>1</em> healthy volunteers [39 +/- <em>7</em> (SD) years] and <em>1</em>8 untreated essential hypertensive subjects [43 +/- 9 years] collected urine for 24 h after a physical examination and laboratory tests. Radioimmunoassay measurements of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] in urine and plasma were done as described previously. Sitting systolic and diastolic blood pressures (+/- SD) averaged <em>1</em><em>1</em>8 +/- <em>1</em><em>1</em>/<em>7</em>4 +/- <em>7</em> mm Hg and <em>1</em>46 +/- <em>1</em>6/96 +/- 8 mm Hg in normal and essential hypertensive subjects, respectively (P < .00<em>1</em>), whereas 24 h urinary volume was not different in normal and essential hypertensive subjects (P>> .05). The concentration of Ang-(<em>1</em>-<em>7</em>) in the urine of normal subjects averaged 62.6 +/- 22.6 pmol/L corresponding to a urinary excretion rate of 98.9 +/- 44.<em>7</em> pmol/24 h. Concurrent measurements of plasma Ang-(<em>1</em>-<em>7</em>) showed that the content of Ang-(<em>1</em>-<em>7</em>) in urine was 2.5-fold higher than that measured in the plasma. In contrast, untreated essential hypertensive subjects had lower concentrations and 24 h urinary excretion rates of Ang-(<em>1</em>-<em>7</em>) averaging 39.4 +/- <em>1</em>8.0 pmol/L and 60.2 +/- <em>1</em>4.6 pmol/24 h, respectively, (P < .00<em>1</em>). Differences in the excretory rate of Ang-(<em>1</em>-<em>7</em>) between normal volunteers and essential hypertensive subjects were not modified by normalization of the data by urinary creatinine excretion rates. Urinary concentrations of Ang-(<em>1</em>-<em>7</em>) correlated inversely with systolic, diastolic and mean arterial pressures (r = -0.48, P < .00<em>1</em>). Both urinary Ang-(<em>1</em>-<em>7</em>) [odds ratio of 0.92 (95% CI: 0.88-0.9<em>7</em>)] and age were independent predictors of systolic blood pressure. These studies demonstrated the presence of Ang-(<em>1</em>-<em>7</em>) in urine and the existence of reduced levels of the heptapeptide in individuals with untreated essential hypertension. The relatively higher concentrations of Ang-(<em>1</em>-<em>7</em>) in urine compared to plasma agrees with data that showed that Ang-(<em>1</em>-<em>7</em>) may contribute to the regulation of blood pressure. The inverse association between Ang-(<em>1</em>-<em>7</em>) and arterial pressure provides a potential marker for the characterization of forms of essential hypertension associated with reduced production or activity of vasodilator hormones.

To investigate the effects of long-term pressure overload on regional myocardial substrate use, we performed quantitative autoradiography using 2-deoxy-D-[U-<em>1</em>4C]glucose (<em>1</em>4C-DG) and beta-methyl[<em>1</em>-<em>1</em>4C]heptadecanoic acid (<em>1</em>4C-BMHDA) in conscious rats with a <em>1</em>0-week ascending aortic constriction. Heart weight/body weight ratio increased by 2<em>7</em>% in aortic-constricted rats as compared with sham-operated rats (p less than 0.0<em>1</em>). Myocardial <em>1</em>4C-DG uptake increased (258 +/- 63 vs. <em>1</em>44 +/- 4<em>1</em> nCi/g, p less than 0.0<em>1</em>, n = 6 for each group); however, <em>1</em>4C-BMHDA extraction decreased (25<em>1</em> +/- 69 vs. 342 +/- <em>7</em>5 nCi/g, p less than 0.05, n = <em>7</em> for each group) in aortic-constricted rats as compared with sham-operated rats. In sham-operated rats, both <em>1</em>4C-DG and <em>1</em>4C-BMHDA uptakes were higher in the left ventricular anterior and lateral walls as compared with the posterior wall or the interventricular septum. In aortic-constricted rats, <em>1</em>4C-DG uptake also increased in the interventricular septum, as well as in the left ventricular anterior and lateral walls, as compared with the posterior wall. There was, however, no regional difference in <em>1</em>4C-BMHDA extraction among these four regions. Myocardial blood flow distribution determined by 4-[N-methyl-<em>1</em>4C]iodoantipyrine or myocyte width showed no regional variations among the four regions, either in aortic-constricted or sham-operated rats. Regional interstitial fibrosis was small in either group. The present study suggests that myocardial substrate uptake is altered nonhomogeneously, and that the nonhomogeneity is not because of regional variations in blood flow distribution, myocyte hypertrophy, or interstitial fibrosis. The results of <em>angiotensin</em> II-induced acute pressure overloading in other sham-operated rats, in which a remarkable increase in myocardial <em>1</em>4C-BMHDA extraction (n = 3, p less than 0.0<em>1</em>) and no difference in <em>1</em>4C-DG uptake (n = 3) as compared with normotensive sham-operated rats were elicited, suggest that the findings in aortic-constricted rats are not direct responses to increased left ventricular pressure itself but rather should be explained by still unknown factors related to prolonged pressure overload.

OBJECTIVE

Determine the effect of a matrix metalloproteinase inhibitor (MMPi) and angiotensin converting enzyme inhibitor (ACEi) on collagen, MMP, tissue inhibitors of MMPs (TIMPs) expression in the spontaneously hypertensive heart failure (SHHF) rat.

METHODS

Six groups were tested: normotensive 9- and 13-month-old Wistar-Furth (WF) rats, 9-month-old SHHFs (compensatory hypertrophy), 13-month-old SHHFs with HF, and 13-month-old SHHFs orally administered with either an MMPi (PD166793, 5 mgkg(-1)day(-1)) or ACEi (quinapril, 10 mgkg(-1)day(-1)) for 4 months. Collagen volume fraction was assessed histomorphometrically. Left ventricular (LV) mRNA [MMP-1,-2,-3,-7,-9,-11,-13,-14; TIMP-1,-2,-3,-4; and collagen alpha1(I) and alpha1(III)] and protein (MMP-2 and MMP-9 zymographic activity; Western blot analysis of MMP-13, and TIMP-1,-2,-4) levels could be quantified.

RESULTS

Collagen mRNA levels were elevated in SHHFs compared to age-matched controls, but collagen volume fraction was elevated only in 13-month-old SHHFs (approximately 2x). Only MMP-2 mRNA levels increased significantly with HF. However, MMP-2 and MMP-9 zymographic activity, and MMP-13 protein levels increased. TIMP-1 and TIMP-2 mRNA and protein levels increased, and TIMP-4 protein levels decreased in SHHFs vs. controls. Both drug treatments reduced LV dilation; preserved systolic function; and normalized MMP/TIMP expression. Both drug treatments also reduced collagen volume fraction, but only quinapril reduced collagen mRNA levels and LV hypertrophy.

CONCLUSIONS

The divergent effect of MMPi and ACEi on collagen mRNA levels and hypertrophy indicate that drug efficacy is mediated by different pathways in the SHHF rat.

Hypertension and diabetes are associated with an increased arterial stiffness. A direct blood pressure-independent effect of <em>angiotensin</em>-converting enzyme inhibitors on arterial stiffness has never been unequivocally demonstrated. In this mechanistic study, we used an experimental design in which patients responding to <em>1</em> month treatment with 4 mg perindopril were randomized double-blind to either 4 mg perindopril or 8 mg perindopril for 6 months. We determined carotid distensibility with echotracking and applanation tonometry at baseline and after the <em>7</em>-month treatment period in 5<em>7</em> essential hypertensive patients with type 2 diabetes (age 63+/-<em>7</em> years). We monitored ambulatory blood pressure at baseline and after treatment. After <em>7</em> months treatment, 24-hour ambulatory blood pressure significantly decreased, with no significant difference between 4 mg and 8 mg perindopril. Carotid distensibility increased more after 8 mg perindopril compared with 4 mg perindopril (8 mg: from <em>1</em>3.<em>1</em>+/-5.9 to <em>1</em>6.0+/-6.<em>7</em> kPa(-<em>1</em>)x<em>1</em>0(-3); 4 mg: from <em>1</em>3.2+/-5.2 to <em>1</em>2.<em>7</em>+/-5.9 kPa(-<em>1</em>)x<em>1</em>0(-3); ANOVA, dose-period interaction, P<0.05). Carotid internal diameter and elastic modulus were significantly lower after 8 mg perindopril compared with 4 mg perindopril, independent of blood pressure reduction. These results indicate a dose-dependent and blood pressure-independent reduction in carotid stiffness under chronic treatment with an <em>angiotensin</em>-converting enzyme inhibitor. They suggest that arterial distensibility was increased through an inward remodeling, leading to a reduction in wall stress, thus reducing elastic modulus. They also suggest that long-term administration of high doses (8 mg) of perindopril is required to improve carotid structure and function in hypertensive patients with type 2 diabetes.

<em>Angiotensin</em> II (Ang II) stimulation of the Ang type <em>1</em> receptor (AT(<em>1</em>)R) facilitates myocardial remodeling through NADPH oxidase-mediated generation of oxidative stress. Components of the renin-<em>angiotensin</em> system constitute an autocrine/paracrine unit in the myocardium, including renin, which is the rate-limiting step in the generation of Ang II. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo renin inhibition and/or AT(<em>1</em>)R blockade in a rodent model of chronically elevated tissue Ang II levels, the transgenic (mRen2)2<em>7</em> rat (Ren2). The Ren2 overexpresses the mouse renin transgene with resultant hypertension, insulin resistance, and cardiovascular damage. Young (6- to <em>7</em>-wk-old) heterozygous (+/-) male Ren2 and age-matched Sprague-Dawley rats were treated with the renin inhibitor aliskiren, which has high preferential affinity for human and mouse renin, an AT(<em>1</em>)R blocker, irbesartan, or placebo for 3 wk. Myocardial NADPH oxidase activity and immunostaining for NADPH oxidase subunits and 3-nitrotyrosine were evaluated and remodeling changes assessed by light and transmission electron microscopy. Blood pressure, myocardial NADPH oxidase activity and subunit immunostaining, 3-nitrotyrosine, perivascular fibrosis, mitochondrial content, and markers of activity were significantly increased in Ren2 compared with SD littermates. Both renin inhibition and blockade of the AT(<em>1</em>)R significantly attenuated cardiac functional and structural alterations, although irbesartan treatment resulted in greater reductions of both blood pressure and markers of oxidative stress. Collectively, these data suggest that both reduce changes driven, in part, by Ang II-mediated increases in NADPH oxidase and, in part, increases in blood pressure.

BACKGROUND

MANTICORE 101 - Breast (Multidisciplinary Approach to Novel Therapies in Cardiology Oncology Research) is a randomized trial to determine if conventional heart failure pharmacotherapy (angiotensin converting enzyme inhibitor or beta-blocker) can prevent trastuzumab-mediated left ventricular remodeling, measured with cardiac MRI, among patients with HER2+ early breast cancer.

METHODS

One hundred and fifty-nine patients with histologically confirmed HER2+ breast cancer will be enrolled in a parallel 3-arm, randomized, placebo controlled, double-blind design. After baseline assessments, participants will be randomized in a 1:1:1 ratio to an angiotensin-converting enzyme inhibitor (perindopril), beta-blocker (bisoprolol), or placebo. Participants will receive drug or placebo for 1 year beginning 7 days before trastuzumab therapy. Dosages for all groups will be systematically up-titrated, as tolerated, at 1 week intervals for a total of 3 weeks. The primary objective of this randomized clinical trial is to determine if conventional heart failure pharmacotherapy can prevent trastuzumab-mediated left ventricular remodeling among patients with HER2+ early breast cancer, as measured by 12 month change in left ventricular end-diastolic volume using cardiac MRI. Secondary objectives include 1) determine the evolution of left ventricular remodeling on cardiac MRI in patients with HER2+ early breast cancer, 2) understand the mechanism of trastuzumab mediated cardiac toxicity by assessing for the presence of myocardial injury and apoptosis on serum biomarkers and cardiac MRI, and 3) correlate cardiac biomarkers of myocyte injury and extra-cellular matrix remodeling with left ventricular remodeling on cardiac MRI in patients with HER2+ early breast cancer.

CONCLUSIONS

Cardiac toxicity as a result of cancer therapies is now recognized as a significant health problem of increasing prevalence. To our knowledge, MANTICORE will be the first randomized trial testing proven heart failure pharmacotherapy in the prevention of trastuzumab-mediated cardiotoxicity. We expect the findings of this trial to provide important evidence in the development of guidelines for preventive therapy.

BACKGROUND

ClinicalTrials.gov: NCT01016886.

The antihypertensive effect of blockade of the brain renin-<em>angiotensin</em> system (brain RAS) was investigated in DOCA (deoxycorticosterone acetate)-salt hypertensive rats. Continuous intracerebroventricular (ICV) administration of SQ<em>1</em>4225 (SQ; <em>1</em>.25 micrograms X 0.5 microliter-<em>1</em> X h-<em>1</em>) for <em>7</em> days attenuated the increase in blood pressure (99 +/- 5 vs. <em>1</em><em>1</em>6 +/- 4 mmHg on the <em>7</em>th day) and also reduced the elevation of blood pressure (<em>1</em>5<em>7</em> +/- <em>7</em> vs. <em>1</em>38 +/- 6 mmHg) in these hypertensive rats. Attenuation of increasing blood pressure in the developing phase following ICV SQ treatment was accompanied by decrease of fluid intake and prevention of elevation of the plasma vasopressin. In the established phase, in addition to reduction of the plasma vasopressin and decrease of fluid intake, restoration of the impaired baroreceptor reflexes was brought about by ICV SQ treatment. These results indicate that the brain RAS strongly influences the regulation of blood pressure in DOCA-salt hypertensive rats and that its mechanism of action is closely related to changes in sodium excretion, vasopressin, and the baroreceptor reflexes.

BACKGROUND

The mitogen-activated protein kinase (MAPK) cascade is an important intracellular mediator of <em>angiotensin</em> II (Ang II)-induced cell growth and differentiation. Here, we examined the effect of <em>angiotensin</em> II type <em>1</em> receptor (AT<em>1</em>) receptor blockade on renal injury and MAPK activity in Dahl salt-sensitive (DS) rats.

METHODS

DS rats were maintained on a high (H: 8.0%NaCl, N= 8) or low (L: 0.3%NaCl, N= 7) salt diet, or H + candesartan cilexetil (<em>1</em>0 to <em>1</em>5 mg/kg/day, N= 8). Urinary protein excretion (UproteinV), renal cortical collagen content, and glomerular injury (assessed by semiquantitative morphometric analysis) were determined after 4-week treatments. Plasma and kidney Ang II levels were measured by radioimmunoassay. Protein levels of AT<em>1</em> and AT2 receptors in the renal cortical tissues were analyzed by Western-blotting analyses. MAPKs activities, including extracellular signal-regulated kinases (ERK)<em>1</em>/2, c-Jun NH2-terminal kinases (JNK), p38 MAPK, and Big-MAPK-<em>1</em> (BMK<em>1</em>), were measured by Western-blotting analyses or in vitro kinase assays.

RESULTS

DS/H rats showed higher mean blood pressure (MBP), UproteinV, and renal cortical collagen content than DS/L rats. Increased ERK<em>1</em>/2, JNK, and BMK<em>1</em> activities were observed in renal cortical tissues of DS/H rats (approximately 6.3-, 4.5-, and 2.5-fold, respectively), whereas p38 MAPK activity was unchanged. Plasma Ang II levels were significantly reduced in DS/H rats compared with DS/L rats, whereas kidney Ang II contents and AT<em>1</em> receptor protein levels were similar. Candesartan did not alter MBP, but significantly reduced UproteinV and collagen content, and ameliorated progressive sclerotic and proliferative glomerular changes. Furthermore, candesartan decreased renal tissue Ang II contents (from 2<em>1</em>6 +/- <em>1</em>9 to 46 +/- 3 fmol/mL) and ERK<em>1</em>/2, JNK, and BMK<em>1</em> activities (-45%, -60%, and -70%, respectively) in DS/H rats.

CONCLUSIONS

In DS hypertensive rats, some of the renoprotective effects of AT<em>1</em> receptor blockade are accompanied by reductions in intrarenal Ang II contents and MAPK activity, which might not be mediated through arterial pressure changes.

BACKGROUND

In the present study, we investigated the possible modulatory effect of losartan, an angiotensin receptor blocker, on oxidative stress induced by cisplatin (CDDP) as well as on CDDP uptake by the kidney.

METHODS

Rats were injected with a single dose of CDDP (7 mg/kg) and/or losartan (in either a single dose of 60 mg/kg or divided doses (10 mg/kg daily for 6 days), starting 1 h before CDDP injection. In addition, rat renal cortical slices were incubated with CDDP (2 mM) and/or losartan (2 mM) for 4 h. Nephrotoxicity was evaluated by measuring serum creatinine and blood urea nitrogen (BUN) in vivo and lactate dehydrogenase (LDH) leakage in vitro; histopathological examination of kidney tissue was also done. Oxidative stress markers including reduced glutathione (GSH) and lipid peroxides were also assessed. Furthermore, CDDP uptake by renal cortical slices was determined.

RESULTS

Losartan has protective effects against CDDP-induced nephrotoxicity as evidenced by restoration of normal serum levels of creatinine and BUN, and LDH leakage. Histopathological examination of the kidney confirmed these results. Also, losartan significantly counteracted CDDP-induced lipid peroxidation and GSH depletion. However, losartan did not affect CDDP uptake by the kidney.

CONCLUSIONS

Our results indicate that losartan has proved to be a promising drug for clinical use as a nephroprotectant against CDDP-induced nephrotoxicity.

The hemodynamic effects of the glucagon-like peptide-<em>1</em> (GLP-<em>1</em>) receptor agonist, exendin-4, and putative underlying mechanisms were assessed in conscious male Sprague-Dawley rats. At a dose of 25 ng kg(-<em>1</em>) i.v., exendin-4 had little effect, but doses of 250 and 2500 ng kg(-<em>1</em>) had significant tachycardic effects (+66 +/- 9 and +95 +/- <em>1</em>6 beats min(-<em>1</em>) at 5 min, respectively) and pressor actions (+<em>1</em>0 +/- 2 and +<em>1</em>2 +/- <em>1</em> mm Hg), accompanied by substantial falls in mesenteric vascular conductance (-38 +/- 3% and -4<em>7</em> +/- 3%) and increases in hindquarters vascular conductance (+82 +/- <em>1</em>4% and +<em>1</em>26 +/- <em>1</em>5%). The latter were likely due to adrenaline-mediated activation of beta(2) adrenoceptors since they were abolished by the beta(2) adrenoceptor antagonist, ICI <em>1</em><em>1</em>855<em>1</em> [(+/-)-<em>1</em>-[2,3-(dihydro-<em>7</em>-methyl-<em>1</em>H-inden-4-yl)oxy]-3-[(<em>1</em>-methylethyl)amino]-2-butanol) hydrochloride], or propranolol [(RS)-<em>1</em>-[(<em>1</em>-methylethyl)amino]-3-(<em>1</em>-naphthalenyloxy)-2-propanol], and absent in adrenal-demedullated rats. In the presence of beta-adrenoceptor antagonism, the tachycardic effects of exendin-4 were suppressed, but the pressor action was enhanced. Enhancement of the pressor action of exendin-4 was not seen in adrenal-demedullated rats or in animals given phentolamine in addition to propranolol, consistent with a component of the pressor action of exendin-4 being due to an adrenaline-mediated positive inotropic effect mediated by alpha-adrenoceptors. The mesenteric vasoconstrictor effect of exendin-4 was unaffected by antagonism of alpha-adrenoceptors, vasopressin receptors, <em>angiotensin</em> receptors, or GLP-<em>1</em> receptors, although antagonism of the latter substantially inhibited the hindquarter vasodilator effects of exendin-4. These results are consistent with exendin-4 having cardiovascular effects through GLP-<em>1</em> receptor-dependent and -independent mechanisms, some of which involve sympathoadrenal activation.

OBJECTIVE

To determine the associations between classes of antihypertensive medication use and the risk of cognitive impairment among elderly hypertensive men.

METHODS

The Honolulu-Asia Aging Study is a prospective, community-based cohort study of Japanese American men conducted in Honolulu, Hawaii. We examined 2,19<em>7</em> participants (mean age <em>7</em><em>7</em> years at cohort entry, 1991-1993, followed through September 2010) with hypertension and without dementia or cognitive impairment at baseline, who provided information on medication use. Cognitive function was assessed at <em>7</em> standardized examinations using the Cognitive Abilities Screening Instrument (CASI). Cognitive impairment was defined as a CASI score (<em>7</em>4.

RESULTS

A total of 854 men developed cognitive impairment (median follow-up, 5.8 years). β-Blocker use as the sole antihypertensive drug at baseline was consistently associated with a lower risk of cognitive impairment (incidence rate ratio [IRR] 0.69; 95% confidence interval [CI] 0.50-0.94), as compared with men not taking any antihypertensive medications, adjusting for multiple potential confounders. The use of diuretics, calcium channel blockers, angiotensin-converting enzyme inhibitors, or vasodilators alone was not significantly associated with cognitive impairment. Results were similar excluding those with cardiovascular disease or <1 year of follow-up, and additionally adjusting for pulse pressure, heart rate, baseline and midlife systolic blood pressure, and midlife antihypertensive treatment (IRR 0.65; 95% CI 0.45-0.94). The association between β-blocker use and cognitive impairment was stronger among men with diabetes, men aged>><em>7</em>5 years, and those with pulse pressure ≥<em>7</em>0 mm Hg.

CONCLUSIONS

β-blocker use is associated with a lower risk of developing cognitive impairment in elderly Japanese American men.

We have recently described, in the mouse aorta, the vasodilator effect of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang-(<em>1</em>-<em>7</em>)) was mediated by activation of the Mas Ang-(<em>1</em>-<em>7</em>) receptor and that A-<em>7</em><em>7</em>9 and D-Pro<em>7</em>-Ang-(<em>1</em>-<em>7</em>) act as Mas receptor antagonists. In this work we show pharmacological evidence for the existence of a different Ang-(<em>1</em>-<em>7</em>) receptor subtype mediating the vasodilator effect of Ang-(<em>1</em>-<em>7</em>) in the aorta from Sprague-Dawley (SD) rats. Ang-(<em>1</em>-<em>7</em>) induced an endothelium-dependent vasodilator effect in aortic rings from SD rats which was inhibited by removal of the endothelium and by L-NAME (<em>1</em>00 microM) but not by indomethacin (<em>1</em>0 microM). The Ang-(<em>1</em>-<em>7</em>) receptor antagonist D-Pro<em>7</em>-Ang-(<em>1</em>-<em>7</em>) (0.<em>1</em> microM) abolished the vasodilator effect of the peptide. However, the other specific Ang-(<em>1</em>-<em>7</em>) receptor antagonist, A-<em>7</em><em>7</em>9 in concentrations up to <em>1</em>0 microM, did not affect vasodilation induced by Ang-(<em>1</em>-<em>7</em>). The Ang II AT<em>1</em> and AT2 receptors antagonists CV<em>1</em><em>1</em>9<em>7</em>4 (0.0<em>1</em> microM) and PD<em>1</em>233<em>1</em>9 (<em>1</em> microM), respectively, the bradykinin B2 receptor antagonist HOE <em>1</em>40 (<em>1</em> microM) and the inhibitor of ACE captopril (<em>1</em>0 microM) did not change the effect of Ang-(<em>1</em>-<em>7</em>). Our results show that in the aorta of SD rats, the vasodilator effect of Ang-(<em>1</em>-<em>7</em>) is dependent on endothelium-derived nitric oxide. This effect is mediated by the activation of Ang-(<em>1</em>-<em>7</em>) receptors sensitive to D-Pro<em>7</em>-Ang-(<em>1</em>-<em>7</em>), but not to A-<em>7</em><em>7</em>9, which suggests the existence of a different Ang-(<em>1</em>-<em>7</em>) receptor subtype.

OBJECTIVE

This study examined the potential role of Angiotensin II for the regulation of angiogenesis associated genes in receptor positive and negative human breast cancer.

METHODS

Expression of different Renin-Angiotensin system (RAS) components in human breast cancer tissue was investigated using immunofluorescence, and in a receptor positive (MCF-7) and receptor negative (MDA-MB 468) breast cancer cell line by performing immunocytochemistry and RT-PCR. Both cell lines were stimulated with Angiotensin II and Angiotensin II receptor type 1 (At(1)R) blocker Candesartan, and gene expression of vascular endothelial growth factor (VEGF), Angiopoietin 1 and 2 (Ang-1 and Ang-2), tissue inhibitor of matrix metalloproteinases 1 (TIMP-1), and hypoxia inducible transcription factor 2alpha (HIF-2alpha) were quantified by TaqMan-Real-Time PCR analysis.

RESULTS

RAS components, Angiotensinogen, Renin, Angiotensin I-converting enzyme (ACE), and At(1)R and At(2)R were expressed in hormone-receptor negative and positive human breast cancer tissue as well as in MDA-MB 468 and in MCF-7 human breast cancer cells. In addition, we found expression of VEGF, Ang-1, TIMP-1, and HIF-2alpha in both cell lines. However, only in receptor negative MDA-MB 468 cells, did Angiotensin II significantly increase gene expression of VEGF, HIF-2alpha, and TIMP-1. This effect was completely inhibited by Candesartan.

CONCLUSIONS

In conclusion, it is hypothesized that Angiotensin II may be involved in regulation of tumor angiogenesis especially in receptor negative breast cancer by regulation of angiogenesis associated genes via At(1)R. These findings are the first evidence for targeting tumor angiogenesis by inhibition of At(1)R in receptor negative human breast cancer cells and may lead to new therapeutical anticancer strategies based upon inhibition of At(1)R.

Accumulation of a large body of evidence during the past two decades testifies to the complexity of the renin-<em>angiotensin</em> system (RAS). The incorporation of novel enzymatic pathways, resulting peptides, and their corresponding receptors into the biochemical cascade of the RAS provides a better understanding of its role in the regulation of cardiovascular and renal function. Hence, in recent years, it became apparent that the balance between the two opposing effector peptides, <em>angiotensin</em> II and <em>angiotensin</em>-(<em>1</em>-<em>7</em>), may have a pivotal role in determining different cardiovascular pathophysiologies. Furthermore, our recent studies provide evidence for the functional relevance of a newly discovered rat peptide, containing two additional amino acid residues compared to <em>angiotensin</em> I, first defined as pro<em>angiotensin</em>-<em>1</em>2 [<em>angiotensin</em>-(<em>1</em>-<em>1</em>2)]. This review focuses on <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and its important contribution to cardiovascular function and growth, while introducing <em>angiotensin</em>-(<em>1</em>-<em>1</em>2) as a potential novel <em>angiotensin</em> precursor.

Abnormal regulation of the renin <em>angiotensin</em> system such as enhanced renal AT<em>1</em>R function and reduced ACE2 activity contributes to obesity-related hypertension. Here, we tested whether long-term AT2R activation affects renal function in obesity using lean and obese Zucker rats treated with the AT2R agonist CGP42<em>1</em><em>1</em>2A for 2 weeks. This caused blood pressure to decrease by <em>1</em>3 mm Hg, which was associated with increased urinary sodium excretion in the obese rats. Cortical ACE2 expression and activity, the Mas receptor (MasR), and its ligand <em>angiotensin</em>-(<em>1</em>-<em>7</em>) were all increased in CGP-treated obese compared with control rats. Candesartan-induced natriuresis, a measure of AT₁R function, was reduced but cortical AT₁R expression and <em>angiotensin</em> II levels were similar in CGP-treated obese compared with control rats. Renin and AT2R expression in obese rats was not affected by CGP treatment. In HK-2 cells in vitro, CGP treatment caused increased ACE2 activity and MasR levels but decreased AT₁R levels and renin activity. Thus, long-term AT2R activation shifts the opposing arms of renin <em>angiotensin</em> system and contributes to natriuresis and blood pressure reduction in obese animals. Our study highlights the importance of AT2R as a target for treating obesity-related hypertension.

OBJECTIVE

The renin-<em>angiotensin</em> system (RAS) plays a critical role in blood pressure control and body fluid and electrolyte homeostasis. In the past few years, <em>angiotensin</em> (Ang) (<em>1</em>-<em>7</em>) has been reported to counteract the effects of Ang II and was even considered as a new therapeutical target in RAS. The present study aimed to investigate the effect of Ang (<em>1</em>-<em>7</em>) administration on a diabetic animal model and the modulation on local RAS.

METHODS

Streptozotocin (STZ) injection-induced diabetic rats were used in the experiment. The animals were divided into 3 groups: (<em>1</em>) control; (2) STZ-induced diabetes; and (3) STZ-induced diabetes with chronic Ang (<em>1</em>-<em>7</em>) treatment [D+Ang(<em>1</em>-<em>7</em>)]. In the D+Ang(<em>1</em>-<em>7</em>) group, a dose of 25 microg x kg(-<em>1</em>) x h(-<em>1</em>) of Ang (<em>1</em>-<em>7</em>) was continually injected through the jugular vein by embedding miniosmotic pump for 6 weeks. Plasma glucose, ratio of kidney to body weight, and 24 h urine protein and serum creatinine were monitored by conventional measurement. Plasma and renal Ang II levels were measured by radioimmunoassay. Ang-converting enzyme (ACE), ACE2, Ang II type <em>1</em> (AT<em>1</em>) receptor, Ang II type 2 (AT2) receptor, Ang (<em>1</em>-<em>7</em>) Mas receptor, and TGF- beta<em>1</em> mRNA levels were measured by real time PCR; ACE, ACE2, and TGF- beta<em>1</em> protein levels were analyzed by Western blotting.

RESULTS

The renal function of diabetic rats was significantly retrogressed when compared with that of control rats. After the treatment by constant Ang (<em>1</em>-<em>7</em>) vein injection for 6 weeks, renal function was found to be even worse than diabetic rats, and both TGF-beta<em>1</em> mRNA and protein levels were elevated in the D+Ang(<em>1</em>-<em>7</em>) group compared with the diabetic rats. The real-time PCR result also showed an increase in ACE mRNA expression and decrease in ACE2 mRNA level in the D+Ang(<em>1</em>-<em>7</em>) group when compared with diabetic rats. The number of AT<em>1</em> receptors increased in the Ang (<em>1</em>-<em>7</em>)-injected group, while the number of AT2 and Mas receptors decreased.

CONCLUSIONS

Exogenous Ang (<em>1</em>-<em>7</em>) injection did not ameliorate STZinduced diabetic rat renal injury; on the contrary, it accelerated the progressive diabetic nephropathies.

The Renin <em>Angiotensin</em> System (RAS) is a pivotal physiological regulator of heart and kidney homeostasis, but also plays an important role in the pathophysiology of heart and kidney diseases. Recently, new components of the RAS have been discovered, including <em>angiotensin</em> converting enzyme 2 (ACE2), <em>Angiotensin</em>(Ang)-(<em>1</em>-<em>7</em>), Mas receptor, Ang-(<em>1</em>-9) and Alamandine. These new components of RAS are formed by the hydrolysis of Ang I and Ang II and, in general, counteract the effects of Ang II. In experimental models of heart and renal diseases, Ang-(<em>1</em>-<em>7</em>), Ang-(<em>1</em>-9) and Alamandine produced vasodilation, inhibition of cell growth, anti-thrombotic, anti-inflammatory and anti-fibrotic effects. Recent pharmacological strategies have been proposed to potentiate the effects or to enhance the formation of Ang-(<em>1</em>-<em>7</em>) and Ang-(<em>1</em>-9), including ACE2 activators, Ang-(<em>1</em>-<em>7</em>) in hydroxypropyl β-cyclodextrin, cyclized form of Ang-(<em>1</em>-<em>7</em>) and nonpeptide synthetic Mas receptor agonists. Here, we review the role and effects of ACE2, ACE2 activators, Ang-(<em>1</em>-<em>7</em>) and synthetic Mas receptor agonists in the control of inflammation and fibrosis in cardiovascular and renal diseases and as counter-regulators of the ACE-Ang II-AT<em>1</em> axis. We briefly comment on the therapeutic potential of the novel members of RAS, Ang-(<em>1</em>-9) and alamandine, and the interactions between classical RAS inhibitors and new players in heart and kidney diseases.

Patients with kidney failure are at high risk of a cardiac death and frequently develop left ventricular hypertrophy (LVH). The mechanisms involved in the cardiac structural changes that occur in kidney failure are yet to be fully delineated. <em>Angiotensin</em>-converting enzyme (ACE) 2 is a newly described enzyme that is expressed in the heart and plays an important role in cardiac function. This study assessed whether ACE2 plays a role in the cardiac remodelling that occurs in experimental acute kidney injury (AKI). Sprague-Dawley rats had sham (control) or subtotal nephrectomy surgery (STNx). Control rats received vehicle (n = <em>1</em>0), and STNx rats received the ACE inhibitor (ACEi) ramipril, <em>1</em> mg kg(-<em>1</em>) day(-<em>1</em>) (n = <em>1</em>5) or vehicle (n = <em>1</em>3) orally for <em>1</em>0 days after surgery. Rats with AKI had polyuria (P < 0.00<em>1</em>), proteinuria (P < 0.00<em>1</em>) and hypertension (P < 0.00<em>1</em>). Cardiac structural changes were present and characterized by LVH (P < 0.00<em>1</em>), fibrosis (P < 0.00<em>1</em>) and increased cardiac brain natriuretic peptide (BNP) mRNA (P < 0.0<em>1</em>). These changes occurred in association with a significant increase in cardiac ACE2 gene expression (P < 0.0<em>1</em>) and ACE2 activity (P < 0.05). Ramipril decreased blood pressure (P < 0.00<em>1</em>), LVH (P < 0.00<em>1</em>), fibrosis (P < 0.0<em>1</em>) and BNP mRNA (P < 0.0<em>1</em>). These changes occurred in association with inhibition of cardiac ACE (P < 0.05) and a reduction in cardiac ACE2 activity (P < 0.0<em>1</em>). These data suggest that AKI, even at <em>1</em>0 days, promotes cardiac injury that is characterized by hypertrophy, fibrosis and increased cardiac ACE2. <em>Angiotensin</em>-converting enzyme 2, by promoting the production of the antifibrotic peptide <em>angiotensin</em>(<em>1</em>-<em>7</em>), may have a cardioprotective role in AKI, particularly since amelioration of adverse cardiac effects with ACE inhibition was associated with normalization of cardiac ACE2 activity.